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首页> 外文期刊>Nucleic Acids Research >Mutational analysis of Escherichia coli DNA ligase identifies amino acids required for nick-ligation in vitro and for in vivo complementation of the growth of yeast cells deleted for CDC9 and LIG4.
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Mutational analysis of Escherichia coli DNA ligase identifies amino acids required for nick-ligation in vitro and for in vivo complementation of the growth of yeast cells deleted for CDC9 and LIG4.

机译：大肠杆菌DNA连接酶的突变分析可确定体外切口连接和CDC9和LIG4缺失的酵母细胞生长的体内互补所需的氨基酸。

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We report that the NAD-dependent Escherichia coli DNA ligase can support the growth of Saccharomyces cerevisiae strains deleted singly for CDC9 or doubly for CDC9 plus LIG4. Alanine-scanning mutagenesis of E.coli DNA ligase led to the identification of seven amino acids (Lys115, Asp117, Asp285, Lys314, Cys408, Cys411 and Cys432) that are essential for nick-joining in vitro and for in vivo complementation in yeast. The K314A mutation uniquely resulted in accumulation of the DNA-adenylate intermediate. Alanine substitutions at five other positions (Glu113, Tyr225, Gln318, Glu319 and Cys426) did not affect in vivo complementation and had either no effect or only a modest effect on nick-joining in vitro. The E113A and Y225A mutations increased the apparent K (m)for NAD (to 45 and 76 microM, respectively) over that of the wild-type E. coli ligase (3 microM). These results are discussed in light of available structural data on the adenylylation domains of ATP- and NAD-dependent ligases. We observed that yeast cells containing only the 298-amino acid Chlorella virus DNA ligase (a 'minimal' eukaryotic ATP-dependent ligase consisting only of the catalytic core domain) are relatively proficient in the repair of DNA damage induced by UV irradiation or treatment with MMS, whereas cells containing only E.coli ligase are defective in DNA repair. This suggests that the structural domains unique to yeast Cdc9p are not essential for mitotic growth, but may facilitate DNA repair.
机译：我们报告说，NAD依赖的大肠杆菌DNA连接酶可以支持酿酒酵母菌株的生长，对于CDC9单个删除或对CDC9加LIG4双重删除。大肠杆菌DNA连接酶的丙氨酸扫描诱变导致鉴定了七个氨基酸（Lys115，Asp117，Asp285，Lys314，Cys408，Cys411和Cys432），这些氨基酸对于在体外进行缺口连接以及在酵母中进行体内互补至关重要。 K314A突变独特地导致DNA腺苷酸中间体的积累。在其他五个位置（Glu113，Tyr225，Gln318，Glu319和Cys426）的丙氨酸取代不影响体内互补，并且对体外切口连接没有影响或只有中等影响。与野生型大肠杆菌连接酶（3 microM）相比，E113A和Y225A突变增加了NAD的表观K（m）（分别为45和76 microM）。根据有关ATP和NAD依赖性连接酶的腺苷酸化域的可用结构数据，讨论了这些结果。我们观察到仅含有298个氨基酸的小球藻病毒DNA连接酶（仅由催化核心结构域组成的“最小”真核ATP依赖性连接酶）的酵母细胞在修复紫外线辐射或用紫外线处理引起的DNA损伤方面相对熟练。 MMS，而仅包含大肠杆菌连接酶的细胞在DNA修复中存在缺陷。这表明酵母Cdc9p特有的结构域对于有丝分裂生长不是必需的，但可能有助于DNA修复。

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